Modulation of Fluorophore Environment in Host Membranes of Varying Charge

The net electrical charge of the biological membrane represents an important parameter in the organization, dynamics and function of the membrane. In this paper, we have characterized the change in the microenvironment experienced by a membrane-bound fluorescent probe when the charge of the phospholipids constituting the host membrane is changed from zwitterionic to cationic with minimal change in the chemical structure of the host lipid. In particular, we have explored the difference in the microenvironment experienced by the fluorescent probe 2-(9-anthroyloxy)stearic acid (2-AS) in model membranes of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) which are otherwise chemically similar, using the wavelength-selective fluorescence approach and other fluorescence parameters. Our results show that the microenvironment experienced by a membrane probe such as 2-AS is different in POPC and EPOPC membranes, as reported by red edge excitation shift (REES) and other fluorescence parameters. The difference in environment encountered by the probe in the two cases could possibly be due to variation in hydration in the two membranes owing to different charges.     

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6 nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.     

Influence of Cholesterol on Molecular Motions in Spin-Labeled Lipid Bilayers Observed by Stimulated ESE

Electron spin echo (ESE) study was performed for spin-labeled lipids 1-palmitoyl-2-stearoyl-(5-d)-sn-glycero-3-phosphocholine in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine phospholipid bilayer. Recently (Isaev and Dzuba in J Phys Chem B 112:13285–13291, 2008), three-pulse stimulated ESE was shown to be sensitive to two types of orientational motion of spin labels in phospholipid bilayers at low temperatures (~100–150 K). The first one is fast stochastic libration, with correlation time on the nanosecond time scale. The second one is slow rotational motion, developing on the accessible for measurements microsecond time scale in a small range of reorientation angles, ~0.1°–1°. These two types of motions may be easily discriminated by dependences of the echo decay rates on the time delays between the pulses. The presence of cholesterol in lipid bilayers is found to suppress remarkably rotational motions, while on the contrary stochastic librations seem to become somewhat enhanced. These results evidence that cholesterol increases the long-time stability of lipid orientations in the bilayer, with simultaneous increase of fast fluctuations of these orientations. The former may be related to the known condensing effect of cholesterol and to raft formations, while the latter to the ordering effect.     

Tracing Lysozyme-Lipid Interactions with Long-Wavelength Squaraine Dyes

The applicability of the two newly commercial available squaraine labels Square-670-NHS and Seta-635-NHS to exploring protein-lipid interactions has been evaluated. The labels were conjugated to lysozyme (Lz) (squaraine-lysozyme conjugates below referred to as Square-670-Lz and Seta-635-Lz), a structurally well-characterized small globular protein displaying the ability to interact both, electrostatically and hydrophobically with lipids. The lipid component of the model systems was represented by lipid vesicles composed of zwitterionic lipids egg yolk phosphatidylcholine (PC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and their mixtures with anionic lipids either beef heart cardiolipin (CL) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), respectively. Fluorescence intensity of Square-670-Lz was found to decrease upon association with lipid bilayer, while the fluorescence intensity of Seta-635-Lz displayed more complex behavior depending on lipid-to-protein molar ratio. Covalent coupling of squaraine labels to lysozyme exerts different influence on the properties of dye-protein conjugate. It was suggested that Square-670-NHS covalent attachment to Lz molecule enhances protein propensity for self-association, while squaraine label Seta-635-NHS is sensitive to different modes of lysozyme-lipid interactions—within the L:P range 6–11, when hydrophobic protein-lipid interactions are predominant, an aggregation of membrane-bound protein molecules takes place, thereby decreasing the fluorescence intensity of Seta-635-Lz. At higher L:P values (from 22 to 148) when electrostatic interactions are enhanced fluorescence intensity of Seta-635-Lz increases with increasing lipid concentrations.     

Computational investigation of lipid hydration water of Lα 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at three hydration levels

Atomistic-scale simulations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer systems were performed at three hydration levels from 12 water per lipid to 41 water per lipid. The structural and dynamical properties of water at each hydration level are the focus of the current study. The water properties are reported as a function of slab position relative to the lipid central plane using a floating slab model. Water properties depend more strongly on the relative slab position to the lipid than on the hydration level of the lipid. Water hydrogen bonds strengthen as water enters into the lipid, reaching a maximum at the phosphorus density maximum. The ratio of lateral and normal diffusion coefficients of water varies as water enters into the lipid and demonstrates an interesting crossover phenomenon at the phosphorus density maximum regardless of the overall hydration level. While most properties do not change beyond the commonly established excess water point, the water diffusion coefficient still increases upon further hydration. We propose a scheme that classifies lipid hydration water into buried water, interface water, and bulk-like water.     

Temperature effect on thin lipid film elasticity and phase separation: insights from Langmuir monolayer and fluorescence microscopy techniques

Langmuir monolayer pressure isotherms and compressibility modulus measurements of phospholipid mixtures in several Langmuir monolayer systems at the air/water interface were investigated in this study. The ultimate aim was to carry out a comparison of the elasticity modulus for monolayers with different mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), in the presence/absence of cholesterol (Chol). In particular, we were able to propose that the leading force beyond the phase separation into liquid expanded (LE-) and liquid condensed (LC-) phases emerges from the increasing barrier to incorporate DOPC molecules into a highly ordered LC-phase. In addition, our findings suggest that DOPC lipid molecules have a priority to incorporate in a disordered LE-phase, while DPPC and eSM prefer the ordered one. Also, Chol seems to split almost equally into both phases, indicating that Chol has no priority for either phase and there are no particular interactions between Chol and saturated lipid molecules.     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

Tilt angle of lipid acyl chains in unilamellar vesicles determined by ellipsometric light scattering

Ellipsometric light scattering (ELS) at room temperature is applied to unilamellar vesicles (~50 nm radius) of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the gel phase and of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the liquid-crystaline phase. A high sensitivity of this technique to the local anisotropy is found. From the resulting local birefringence, a lower limit of (29 ±0.5)^○ for the average tilt angle of the lipid chains of DPPC with respect to the membrane normal is estimated. This tilt angle value is slightly lower than literature values for the tilt angle in oriented lipid multi-bilayers on solid substrates.     

Transfer of pyrene-dietherphosphatidylcholine to serum lipoproteins

The nonhydrolyzable fluorescent diether analog of phosphatidylcholine, 1-O-hexadecyl-2-0-pyrenedecyl-sn-glycero-3-phosphocholine, has been synthesized as a stable probe for the determination of phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human serum with the new probe at 37°C for 3 hours a characteristic partition equilibrium between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a marker for choline glycerophospholipid distribution between and transport to serum lipoproteins.     

Visualization of lipid-receptor interactions on single cells by time-resolved imaging fluorescence microscopy

The physical interaction between plasma-membrane lipids and the epidermal growth factor (EGF)-receptor was investigated on single A431 human epidermoid carcinoma cells by monitoring fluorescence resonance energy transfer (FRET) between exogeneously added fluorescein-EGF (donor) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC, acceptor) using donor-photobleaching FRET-microscopy. The measured mean FRET-efficiency of 13% is indicative of such a physical interaction and exemplifies the great potential and sensitivity of time-resolved imaging fluorescence microscopy techniques for the study of lipid-receptor interactions on single cells.     

X- and Q-Band Electron Spin Echo Study of Stochastic Molecular Librations of Spin Labels in Lipid Bilayers

Electron spin echo (ESE) of nitroxide spin labels allows detecting fast nanosecond stochastic restricted rotations (stochastic molecular librations), which is a common property of molecules in disordered media including biological systems. Under the typical experimental conditions, the anisotropic electron paramagnetic resonance (EPR) spectrum of a nitroxide is only partly excited by microwave pulses, which allows selecting an anisotropic contribution to the transverse spin relaxation by comparing echo decays at different spectral positions. On the other hand, for low-amplitude orientational motion, the excitation bandwidth is large enough to cover the range of spectral diffusion occurring during the echo formation. To verify that the two-pulse echo decay is indeed related to fast motions, the stimulated electron spin echo can be used. In addition, theory predicts an increase of the relaxation rates at higher microwave resonance frequency. To check this prediction, in the present work we performed a comparative study of ESE decays at microwave X- and Q-bands, for spin-labeled lipids in the gel phase of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. A good agreement found between experimental data and computer simulation provides additional justification for the model of fast stochastic molecular librations.     

Distribution of merocyanine 540 in phospholipid membranes

The interaction of the fluorescent photosensitizer merocyanine 540 (MC-540) with model phospholipid membranes was studied. Two different-colored species (monomers and dimers) of MC-540 were registered in phospholipid liposomes. Variations in both phospholipid composition (DMPC, DPPC, POPC, egg PC) and temperature (15–60°C) resulted in changes in the MC-540 monomerdimer distribution. The values of the monomer-dimer equilibrium constant of MC-540 in egg PC (K=14.8 M), in POPC (K=26.7 M), and in DMPC (K=271.0 M) were determined at the temperature of 23±2°C. Suppression of MC-540 association with phospholipid bilayers was provoked by the addition of albumin to a liposome suspension. Albumin was observed to compete very successfully with lecithins containing saturated fatty acid chains (DPPC, DMPC), while only a weak competition of albumin with unsaturated lecithins (POPC, egg PC) for binding MC-540 molecules was registered.     

Molecular Motion in Frozen Phospholipid Bilayers in the Presence of Sucrose and Sorbitol Studied by the Spin-Echo EPR of Spin Labels

Electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), was applied to spin-labeled stearic acids in phospholipid bilayers hydrated in the presence of sucrose and sorbitol, which are known for their cryoprotective action on biological membranes. The phospholipids were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Stearic acids were labeled by nitroxide 4,4-dimethyl-oxazolidine-1-oxyl (DOXYL) attached rigidly at either the 5th or 16th specific carbon positions. ESE detects fast stochastic small-angle restricted molecular rotations (stochastic molecular librations) with correlation times on the nanosecond timescale. These motions are believed to have the same nature as the anharmonic motions of hydrogen atoms in biological substances detected by neutron scattering and Mössbauer spectroscopy, which become active above 200 K. To ensure that the echo decays indeed originate from fast stochastic molecular librations, a three-pulse stimulated spin echo was employed. It was found that the presence of sucrose or sorbitol suppresses the observed molecular motions. The observed effect was nearly the same for both label positions, indicating that the motions are similarly suppressed near the bilayer surface and in the bilayer interior. This finding suggests non-specific interactions of sugars with bilayer surface, which are likely to influence only the bulk physical properties of hydrated membranes. The results obtained show the usefulness of spin-echo EPR of spin labels when applied to investigate the molecular mechanisms of action of cryoprotective agents on biological systems.     

Influence of (phospho)lipases on properties of mica supported phospholipid layers

The effect of enzymes: lipase from Candida cylindracea (L_Cc), phospholipase A₂ from hog pancreas (PLA₂) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.     

Photoinduced flavin-tryptophan electron transfer across vesicle membranes generates magnetic field sensitive radical pairs

ABSTRACT

Due to the photobiology of the flavoproteins DNA photolyase and cryptochrome, electron transfer reactions between flavins and tryptophan are of significant biological relevance. In addition, electron transfer across vesicle membranes has also seen much attention. In this work, we study the electron transfer reaction between flavins and tryptophan across lipid bilayer membranes in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine small unilamellar vesicles using time-resolved optical absorption microspectroscopy and magnetically affected reaction yield spectroscopy. We demonstrate that riboflavin tetrabutyrate is embedded in the vesicle bilayer and can undergo electron transfer with tryptophan molecules in either the inner water pool or the bulk solution. Remarkably, flavin mononucleotide encapsulated in the inner water pool can undergo electron transfer across the vesicle bilayer to generate a magnetically sensitive radical pair with tryptophan molecules located in the bulk solution. The observed kinetics suggest that back electron transfer occurs between radical pairs generated by diffusive reencounter, either in the vesicle surface water or via electron hopping through degenerate electron exchange.     

Incorporation of peptides in phospholipid aggregates using ultrasound

This study presents the highlights of ultrasonic effects on peptides incorporated on phospholipid aggregates (liposomes). These liposomes or vesicles are known as transport agents in skin drug delivery and for hair treatment. They might be a good model to deliver larger peptides into hair to restore fibre strength after hair coloration, modelling, permanent wave and/or straightening. The preparation of liposomes 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) with peptides (LLLLK LLLLK LLLLK LLLLK; LLLLL LCLCL LLKAK AK) was made by the thin film hydration method. The LUVs (uni-lamellar vesicles) were obtained by sonication, applying different experimental conditions, such as depth (mm) and power intensity (%). Photon-correlation spectroscopy (PCS) and electronic microscopy (EM) results confirmed that the incorporation of these peptides, with different sequence of amino acids, presented differences on the diameter, zeta-potential of membrane surface and shape of liposomes. The liposomes that included peptide LLLLK LLLLK LLLLK LLLLK present an increased in zeta-potential values after using ultrasound and an "amorphous" aspect. Conversely, the liposomes that incorporated the peptide LLLLL LCLCL LLKAK AK presented a define shape (rod shape) and the potential surface of liposome did not change significantly by the use of ultrasound.     

Low-Temperature Dynamical Transition in Lipid Bilayers Detected by Spin-Label ESE Spectroscopy

Data on neutron scattering in biological systems show low-temperature dynamical transition between 170 and 230 K manifesting itself as a drastic increase of the atomic mean-squared displacement, 〈x²〉, detected for hydrogen atoms in the nano- to picosecond time scale. For spin-labeled systems, electron spin echo (ESE) spectroscopy—a pulsed version of electron paramagnetic resonance—is also capable of detection of dynamical transition. A two-pulse ESE decay in frozen matrixes is induced by spin relaxation arising from stochastic molecular librations, and allows to obtain the 〈α²〉τ_c parameter, where 〈α²〉 is a mean-squared angular amplitude of the motion and τ_c is the correlation time lying in the sub- and nanosecond time ranges. In this work, the ESE technique was applied to spin-labeled amphiphilic molecules of three different kinds embedded in bilayers of fully saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Two-pulse ESE data revealed the appearance of stochastic librations above 130 K, with the parameter 〈α²〉τ_c obeying the Arrhenius type of temperature dependence and increasing remarkably above 170–180 K. A comparison with a dry sample suggests that onset of motions is not related with lipid internal motions. Three-pulse ESE experiments (resulting in stimulated echos) in DPPC bilayers showed the appearance of slow molecular rotations above 170–180 K. For D₂O-hydrated bilayers, ESE envelope modulation experiments indicate that isotropic water molecular motions in the nearest hydration shell of the bilayer appear with a rate of ~?10⁵ s^?1 in the narrow temperature range between 175 and 179 K. The similarity of the experimental data found for three different spin-labeled compounds suggests a cooperative character for the ESE-detected molecular motions. The data were interpreted within a model suggesting that dynamical transition is related with overcoming barriers, of 10–20 kJ/mol height, existing in the system for the molecular reorientations.     

Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR

High-field W-band (95 GHz) electron paramagnetic resonance (EPR) study of partitioning of a small nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in multilamellar liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is described. The high-resolution spectra with a high signal-to-noise ratio were combined with automated least-squares simulation analysis to derive accurate partitioning coefficients of TEMPO in the membrane lipid phase and to follow the membrane phase transitions. The isotropic magnetic parameters, g_iso and A_iso were used to characterize the average polarity the spin label is experiencing in the membrane. We also report an empirical correlation between g_iso and A_iso for a set of protic and aprotic solvents and use this correlation to assign domains formed by interdigitation of DPPC bilayer under a high ethanol concentration of 1.2 M.     

Low-Temperature Molecular Motions in Phospholipid Bilayers in Presence of Glycerol as Studied by Spin-Echo EPR of Spin Labels

Glycerol is used as a cryoprotective agent to protect biological systems under freezing conditions. Electron spin echo (ESE) spectroscopy, a pulsed version of EPR, is capable of studying low-temperature molecular motions of nitroxide spin labels. ESE technique was applied to study molecular motions in phospholipid bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with added spin-labeled lipids 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (n-PCSL, n was optionally 5 or 16). Bilayers were hydrated (solvated) either in pure water or in a 1:1 v/v water–glycerol mixture. In the used ESE approach, there were studied stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations). The anisotropic contribution to the echo decay rate, W _anis, was measured, which is proportional, according to theory, to the product of the mean-squared angular amplitude \(\langle \alpha^{ 2} \rangle\) and the correlation time τ _c. W _anis was found to be small below and to sharply increase above 200 K, for the both types of solvents and the both label positions. As compared with hydration by pure water, in presence of glycerol W _anis was larger for the 5th label position while for the 16th one it did not change. Also, for the 5th label position W _anis values were found to be nearly the same as those for a polar spin probe 3,4-dicarboxy-PROXYL which was separately added to the bilayer as a reference and which is assumed to be partitioned only into the solvating shell. These results indicate that motions at the surface of bilayer are governed by the motion of solvating shell while motions in the bilayer interior occur independently. The relation of the obtained data with the dynamical transition phenomenon that is known for biological substances near 200 K from neutron scattering and Mössbauer absorption is discussed.     

Structural Transition in the Micellar Assembly: A Fluorescence Study

Structural transition can be induced in charged micelles by increasing the ionic strength of the medium. Thus, spherical micelles of sodium dodecyl sulfate (SDS) that exist in water at concentrations higher than the critical micelle concentration assume an elongated rod-like structure in the presence of an increased electrolyte concentration. This is known as sphere-to-rod transition. In this paper, we characterize the change in organization and dynamics that is accompanied by the salt-induced sphere-to-rod transition in SDS micelles using wavelength-selective fluorescence and other steady-state and time-resolved fluorescence parameters. Since the change in micelle organization during such structural transition may not be limited to one region of the micelle, we have selectively introduced fluorophores in two distinct regions of the micelle. We used two probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (NBD-cholesterol), for monitoring the two regions of the micelle. NBD-PE monitors the interfacial region of the micellar assembly, while NBD-cholesterol acts as a reporter for the deeper regions of the micellar interior. Our results show that wavelength-selective fluorescence, in combination with other fluorescence parameters, offers a powerful way to monitor structural transitions induced in charged micelles. These results could be significant to changes in membrane morphology observed under certain physiological conditions.